Novel targeted approaches for the treatment of various cancer types are urgently needed. Oncolytic viruses hold promise for the treatment of cancers, because they can target cancer cells without harming normal cells. Among them, two groups of viruses attract much attention as alternative antineoplastic agents: the adenoviruses and the autonomous parvoviruses. Adenoviruses (Ad) have been engineered to function as vectors for delivering therapeutic genes for gene therapy, and as direct cytotoxic agents for oncolytic viral therapy. Rodent autonomous parvoviruses (PVs), on the other hand, show oncolytic and oncosupressive properties and are non-pathogenic for humans.
Adenoviruses (Ads) are non-enveloped, icosahedral, double-stranded DNA viruses. As of today, over 50 different human serotypes have been described with most of them infecting the respiratory or gastrointestinal tracts and the eye [1]. Ad infections are very common and generally not associated with any serious pathogenicity. The Ad genome comprises 30-38 kbp, and is delivered to the nucleus of infected host cells. Ads represent the most popular gene therapy vectors, and were used in about 25% of approved phases I to III clinical trials for vaccine and therapeutic gene transfer during the last 2 decades [2, 3]. This is largely due to the ability of these vectors to efficiently deliver trangenes to a wide range of different cell types [1]. Furthermore, Ads are very versatile tools with remarkable DNA packaging capacity, offering a plethora of possibilities for genetic manipulations. The Ad genome can be modified in different ways in order to restrict its replication or expression to specific tumour cells [4]. Furthermore, it is possible to re-direct Ad entry and render it more specific for cancer cells, through the use of molecular adaptors or genetic engineering of Ad capsid [5, 6]. In addition, Ads can be produced at high titers and quality under good manufacturing practice conditions [6]. As a result, ONYX-015, a hybrid of virus serotype Ad2 and Ad5 with deletions in the E1B-55K and E3B regions was the first engineered replication-selective virus to be used in clinical trials against various tumour entities and a modification of this vector, the E1B-55K deleted adenovirus H101, received marketing approval in China in 2005 for the treatment of head and neck cancer [7, 8].
Autonomous rodent parvoviruses (PVs) are small icosahedral, non-enveloped single-stranded DNA viruses. Their genome is about 5.1 kb long and contains two promoters, P4 and P38, that control the expression of the non-structural (NS1 and NS2) and structural (VP1 and VP2) proteins, respectively [9]. Several PVs, including the minute virus of mice (MVM) and the rat H-1PV, have been shown to be oncolytic and oncosuppressive in various cellular and animal cancer models [10]. Additionally, PVs are non-pathogenic and show low prevalence in humans, favoring their use as therapeutics [11]. H-1PV will be evaluated shortly in a phase I/IIa clinical trial for the treatment of patients with recurrent glioblastoma multiforme [10]. The antineoplastic property of these PVs is due, at least in part, to preferential viral DNA replication and gene expression in malignant cells. This could be traced back to the dependence of parvoviruses on S-phase, and more particularly on cellular factors such as E2F, CREB, ATF and cyclin A which are known to be overexpressed in cancer cells [10]. In addition PVs may take also advantage of the common inability of malignant cells to mount an efficient anti-viral innate immune response [12]. It has been shown that PVs have the ability to induce cell cycle arrest [13] and different death pathways in cancer cells, including necrosis [14], apoptosis [13, 15] and lysosomal dependent cell death [16]. NS1 is the major effector of parvovirus oncotoxicity [13]. Although preclinical studies highlight the anticancer potential of PVs, this feature should be further reinforced in view of the clinical application of these agents. One major limitation lies in the fact that PVs bind and enter into a variety of normal human cells, resulting in the sequestration of a large portion of the administered viral dose away from the tumor target cells. Retargeting PV entry to tumor cells would thus increase the efficacy of PV-based treatments and provide additional safety against eventual side-effects on normal tissues. Furthermore, replication-competent PVs have a limited capacity for accommodating transgenes and tolerate only the insertion of a short transgene (300 bp maximum), thereby hampering strategies to reinforce the anticancer efficacy of PVs by arming them with therapeutic transgenes. It should be also stated that large scale production of PVs, as required for clinical applications, remains a major limitation.
The development of an Ad hybrid vector harboring only part of the parvovirus genome (a parvovirus gene expressing cassette including the P4-NS1-P38 region) and not the whole parvovirus genome has been previously described [36]. For instance, the hybrid described in [36] lacks the ITR regions including NS1-specific nicking sites which are essential for NS1-mediated excision and release of the PV genome from the adenovirus backbone in target cancer cells. It also misses the VP gene precluding the possibility to generate fully infectious parvovirus particles.
An Ad5 adenovirus comprising an AAV genome having the Rep genes controlled by a Tet dependent repressor has been previously described [37]. AAV are dependoviruses and although they belong to the Parvoviridae family they show remarkable differences compared to parvoviruses and are well distinguished viruses therefrom. Most importantly, AAVs depend on a helper virus such as Adenovirus for efficient replication while parvoviruses such as H-1PV are replication competent.
Thus, the problem on which the present invention is based was to provide a means (a) for increasing the efficiency of parvovirus production, (b) for increasing the specificity to cancer cells, and (c) for overcoming the current limitations regarding the insertion/expression of therapeutic transgenes that could complement and reinforce PV-antitumour activities.
This technical problem is solved by providing the embodiments characterized in the claims.
Since the early 1970s, DNA recombinant technology made it possible genetic engineering of a variety of viral vectors, in order to match their needs [17]. In particular, viral chimeras were generated both to analyze the parental viruses [18] and to obtain novel artificial virions that combined the desired properties of the parental viruses and compensate for some of their current limitations [18-25]. In the experiments resulting in the present invention an adeno-parvovirus (Ad-PV) chimera was created by inserting the complete genome of hH-1 PV into the Ad5 genome deleted of the E1 and E3 regions (Ad5ΔE1ΔE3) expecting that this chimera may enhance PV replication in cancer cells through the concomitant expression of Ad helper functions [26-28]. Longer-term benefits for PV-based cancer therapy may also include: (i) the specific delivery of PV genomes to cancer cells by means of retargeted Ads and (ii) Ad genome arming with therapeutic transgenes that potentiate the PV killing activity. Would an adenoviral vector endowed with oncolytic properties be used, the PV component of the chimera should reinforce this antineoplastic activity by (i) expressing the cytotoxic NS1 protein under its natural PV promoter, and (ii) amplifying the antitumour effect through PV excision from the vector, autonomous replication and spreading through the tumor.
The first attempts to develop Ad-PV chimeras failed at the production stage due to the strong negative interference of PV non-structural (NS) proteins, with hybrid vector replication. Thus, a strategy for tightly controlling the expression of the viral NS proteins during the chimera production process was devised. To this end, first the H-1PV-TO parvovirus was engineered, in which the early P4 promoter, controlling the expression of the NS gene unit, was modified by inserting tetracycline operator (TO) elements [29, 30]. In HEK T-REx™-293 cells, which constitutively express the Tet repressor (TetR), the activity of P4-TO was completely inhibited, and consequently neither expression of NS1 protein nor PV replication was detected unless the tetracycline analogous, doxycycline (dox) was added to the medium. On the contrary, in cancer target cells, which do not express TetR, the P4 was fully functional to wild-type levels. Based on these results, the PV-TO genome was inserted into the Ad DNA backbone, generating the Ad-PV-TO chimera. In keeping with above results, the blockage of NS expression, allowed the chimera to be produced at high titers in T-REx™-293 cells. The Ad-PV-TO chimera proved able to efficiently deliver its PV component to cancer cells in which the parvoviral genome was excised from the vector and replicated autonomously, yielding progeny PV particles. Most remarkably the Ad-PV-TO was more efficient in killing various cancer cell lines than the parental PV or Ad (used alone or in combination).
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.